Characterization of intermediate steps in amyloid beta (Aβ) production under near-native conditions.

Abstract

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aβ peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aβ and in particular Aβ42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aβ production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aβ40 and Aβ42 and an increase in shorter Aβ peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aβ51 to Aβ31/Aβ30 and from Aβ49 to Aβ30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aβ48 to Aβ38 compared with Aβ49 to Aβ37. These findings correlate with an increase in the Aβ42/40 ratio. GSMs caused a decrease in Aβ40 and Aβ42 and an increase in Aβ37 and Aβ38 paralleled by an increase of the intermediates Aβ40-38 and Aβ42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aβ production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aβ generation.

De novo Aβ production in membrane preparations from HEK/APPswe cells mimics cellular Aβ production.A, schematic depiction of the APP transmembrane sequence with γ-secretase cleavage sites (γ, ζ, ϵ) and the FAD APP mutations (T714I, V717F, L723P) explored in this study highlighted. B, HEK/APPswe cells were either left non-treated (−) or exposed to the γ-secretase inhibitor (GSI) semagacestat (+) for 19 h prior to cell harvesting and membrane preparation (Substrate accumulation). Exposure of cells with GSI prior to membrane preparation results in higher Aβ1–42 and Aβx-42 signals as compared with membranes derived from non-GSI-treated cells. Direct addition of semagacestat to the membranes in the novo Aβ production assay inhibited Aβ production (Semagacestat (1 μm)). The amount of Aβx-42 is higher than Aβ1–42 suggesting that C83 is the major substrate for the Aβ42 signal detected. C, Western blot analysis of APP derivatives in the same assay reveals AICD production (lane Vehicle). The addition of GSI (semagacestat, 1 μm) to the reactions prevents the formation of AICD (lane GSI). C83 is the dominating APP species in the membranes in accordance with the data obtained in B. Recombinant C99 was loaded as reference (lane C99). D, MSD analysis of different Aβ peptides in HEK/APPswe cell culture supernatants (dark bars) and in the de novo Aβ production assay (light bars), respectively. Data are presented as the relative amount specific Aβ peptides compared with total Aβ and the data are based on three independent experiments. The graph shows the average +S.D., and statistics were calculated with Student's unpaired t test. ns, non significant, **, p < 0.01, ***, p < 0.001. E, raw data from MSD analysis of Aβ peptides from the membrane de novo Aβ assay. Max, no treatment; Min, in presence of 1 μm semagacestat. Data shows the mean of duplicates from three independent experiments (Signal) and the standard deviation (Stdev).

Aβ42 is generated from different APP-derived intermediates. LC-MS analyses of native APP processing in the de novo Aβ production assay in HEK/APPswe-derived membranes. A, total ion chromatogram from LC-MS analysis of APP transmembrane sequence derived peptides in the de novo Aβ production assay. B, settings of the mass spectrometer for the specific detection of the peptides analyzed. C, illustration of γ-secretase dependence of selected peptides. Upper trace: Max signal, no treatment; Lower trace: membrane assay performed in presence of 1 μm of the GSI semagacestat. The scale on the upper and lower traces is the same for each pair of chromatograms and the max signal was set to 100%. Note that the LVM signal (bottom right graphs) was only partially inhibited by semagacestat, which precluded absolute quantification of this peptide. D, graph shows the average amount of γ-secretase activity-dependent peptides and stem from the same three experiments as in D. The data are presented in 3 sets: the Aβ40 and 42 product lines and other peptides, respectively. E, summary of the data presented in an illustration of the APP transmembrane sequence presented as an α-helix in a two-dimensional lay-out. The numbering is according to Aβ1-x. Each peptide is illustrated as an arrow, where black arrows are 3-mer; white arrows are 4-mer; gray arrow is 6-mer. The arrows are written in C->N-terminal direction. Impurities were migrating at the same retention time as peptides LVM, VIA, and VVI and excluded their direct quantification (marked with an asterisk). Statistics were calculated with Student's unpaired t test. ns, nonsignificant, **, p < 0.01, ***, p < 0.001. Some peptides were clearly γ-secretase dependent but we could not perform statistics (na, not applicable), since their concentration was below the limit of quantification (0.2 nm) in the presence of semagacestat. Peptides analyzed but not detected are illustrated as hatched arrows (light gray is 3- and 4-mers; dark gray, 5- to 7-mers).

APP FAD mutants affect APP transmembrane sequence product-line preferences resulting in an increased Aβ42/40 production ratio.A, membranes were prepared from SHSY5Y cells expressing either wild type or the APP FAD mutants illustrated in A. The graph shows the relative level of different Aβ peptides generated in the Immuno/LC-MS assay as determined by MSD analysis. Bars show the average from three independent experiments. B, each APP FAD mutant give rise to an increased Aβ42/40 production ratio as compared with wild-type APP. Note the large effect of the T714I mutant. C, LC-MS analysis of the effect of the APP FAD mutations on APP TMS processing. γ-Secretase-mediated processing of wild-type APP results in a similar pattern of APP transmembrane sequence derivatives as APP processing in membranes derived from non-neuronal membranes (i.e. , D–E). All mutations cause a decrease in the Aβ40 product line and a concomitant retained or increase in the Aβ42 product line. D, two-dimensional illustration of the APP transmembrane sequence with the major reactions affected by the APP FAD mutants highlighted with circles: black circle, retained or increased, gray circle, decreased or retained. All data are from three independent experiments, and the figure shows the average +S.D. Statistics were calculated with Student's unpaired t test. ns, nonsignificant, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

GSMs affect distinct γ-secretase reactions resulting in Aβ modulation. MSD analysis of different P3 peptides in conditioned media from BACE-deficient MEFs cells, exposed to the GSMs AZ4126 A) or R-Flurbiprofen B) for 24 h prior to analyzes. Both compounds show a GSM characteristic modulation of P3 peptides as analyzed by the MSD triplex (Aβ38/40/42) assay using 4G8 as detection antibody. C—F, membranes from HEK/APPswe cells were used in the de novo Aβ production assay and incubated with GSMs R-Flurbiprofen (1 mm), Sulindac Sulfide (125 μm), AZ4800 (50 nm and 25 μm), AZ4126 (50 nm), AZ1136 (25 μm), and E-2012 (5 μm). C, all GSMs cause an Aβ modulation that mimick their effect in cellular assays. D, total Aβ was not affected by any treatment or compound besides AZ4800 at 25 μm, which caused a 25% decrease in Aβ suggesting that the compound becomes inhibitory at higher concentrations. E, same reactions were analyzed by LC-MS analysis. Note the GSM-induced effect on the Aβ40–38, Aβ40–35, Aβ42–39, Aβ38–35, and Aβ46–43 peptides, as well as the opposite effect of first generation GSMs (R-Flurbiprofen and Sulindac Sulfide) and second generation GSMs on Aβ46–43 and Aβ40–35, respectively. The basal level of Aβ49–46 is very low resulting in large experimental variations. The inset shows a chromatogram for Aβ46–43 in the presence of increasing concentrations of AZ4800. AZ4800 (25 μm) was set to 100%. F, ratio of peptides resulting in Aβ42 production/turnover and Aβ40 production/turnover is decreased in presence of the GSMs, and the difference in ratio is highest for Aβ42 for all compounds tested. The figure shows the average result from three independent experiments and the statistics were calculated with Student's unpaired t test. ns, nonsignificant, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Summary of major APP transmembrane sequence processing events and GSM pharmacology. APP is subjected to two major γ-secretase-catalyzed processing routes resulting in Aβ40 and 42, respectively. Minor cleavage events result in cross-talks between the major routes and alternative pathways to modulate Aβ42 production and the Aβ42/40 production ratio (hatched arrows). The major processing routes converge at Aβ34, which is further hydrolyzed by γ-secretase resulting in Aβ30, which is the shortest BACE and γ-secretase-dependent Aβ1-x peptide identified in human CSF (). All peptides (arrows) are written in CàN-terminal direction. Each Aβ peptide may stem from a 3- or 4- (or in case of Aβ34 a 6-) amino acid longer Aβ peptide in a precursor-product cascade. Alternatively, each Aβ peptide is not necessarily derived from a 3- or 4- amino acid longer Aβ peptide. Our data did not directly assess these two alternatives, but the data obtained with GSMs support the former mechanism: GSMs cause an increased turnover (+) of Aβ40 to Aβ37, Aβ42 to Aβ38, and Aβ38 to Aβ34 without affecting either Aβ40 or Aβ42 production to a major extent. All potent non-acid second generation GSMs display a robust increase (+) in Aβ46–43 and a decrease (−) in Aβ40 to Aβ34, whereas two GSMs of the NSAID class display a reduction in Aβ46–43 and a tendency toward increased Aβ40–35 production. Other cleavage events were also affected by some of the GSMs, but to a lesser extent and/or less consistent to the reactions outlined above.